Currently, there are no pharmacological treatments for cocaine addiction partly because the mechanism of cocaine addiction is not clear. Cocaine has 3 high affinity targets in the brain, dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). Cocaine binds and blocks the uptake functions of these transporters, resulting in prolonged and elevated transmitter levels in the synapses, which are believed to underlie the stimulating and rewarding effects of cocaine. Surprisingly, knockout mice with DAT, NET, or SERT individually disrupted still exhibit cocaine reward, suggesting that no single transporter is explicitly required for cocaine reward and drugs preventing cocaine inhibition of a single transporter may not be effective. However, compensatory changes in the knockout mice may have altered the reward pathway. To avoid the compensatory changes, we have generated a knock-in mouse line carrying a DAT mutant that retains the uptake function but is insensitive to cocaine inhibition (DAT-KI mice). In these mice, normal doses of cocaine no longer block DAT, stimulate locomotion, or produce reward. Our results indicate that DAT blockade is required for cocaine reward in mice with a functional DAT. Most significantly, it suggests drugs that antagonize cocaine inhibition of DAT should be effective in blocking cocaine reward. DAT-KI mice provide a unique novel tool to investigate the mechanism of the complex cocaine effects. We propose to continue our current studies. We will examine cocaine responses by DAT-KI mice in several other behavioral tests to dissect out the contribution of DAT in the complex effects of cocaine. In addition, we will use AAV vector to re-introduce wild type DAT back into selected brain regions of DAT-KI mice to restore cocaine-induced DA elevation in those selected regions only and study what cocaine responses are restored. This unique approach allows us to correlate cocaine actions in specific brain regions to specific cocaine effects. Our preliminary data show that we are now able to inject AAV in confined brain regions and groups of mice with varying AAV injections restored cocaine responses in either conditioned place preference test only, locomotor stimulation only, both tests, or none of the tests, demonstrating the feasibility of our approach. The success of the proposed project will significantly enhance our understanding on how cocaine produces its complex effects, which are crucial for our efforts in the development of effective treatment for cocaine addiction. PUBLIC HEALTH RELEVANCE: In the proposed study, we will use molecular, genetic, biochemical and behavioral analysis tools to understand the molecular mechanisms of drug addiction. The success of the proposed project will significantly enhance our understanding on how cocaine produces its complex effects, which are crucial for our efforts in the development of effective treatment for cocaine addiction.